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Plants depend on various cell surface receptors to integrate extracellular signals with developmental programs. One of the best-studied receptors is BRASSINOSTEROID INSENSITIVE 1 (BRI1) in Arabidopsis (Arabidopsis thaliana). Upon binding of its hormone ligands, BRI1 forms a complex with a shape-complementary coreceptor and initiates a signal transduction cascade, which leads to a variety of responses. At the macroscopic level, brassinosteroid (BR) biosynthetic and receptor mutants have similar growth defects, which initially led to the assumption that the signaling pathways were largely linear. However, recent evidence suggests that BR signaling is interconnected with several other pathways through various mechanisms. We recently described that feedback from the cell wall is integrated at the level of the receptor complex through interaction with RECEPTOR-LIKE PROTEIN 44 (RLP44). Moreover, BRI1 is required for another function of RLP44: the control of procambial cell fate. Here, we report a BRI1 mutant, bri1(cnu4), which differentially affects canonical BR signaling and RLP44 function in the vasculature. Although BR signaling is only mildly impaired, bri1(cnu4) mutants show ectopic xylem in place of procambium. Mechanistically, this is explained by an increased association between RLP44 and the mutated BRI1 protein, which prevents the former from acting in vascular cell fate maintenance. Consistent with this, the mild BR response phenotype of bri1(cnu4) is a recessive trait, whereas the RLP44-mediated xylem phenotype is semidominant. Our results highlight the complexity of plant plasma membrane receptor function and provide a tool to dissect BR signaling-related roles of BRI1 from its noncanonical functions.

The plant steroid hormones brassinosteroids (BRs) regulate growth in part through altering the properties of the cell wall, the extracellular matrix of plant cells. Conversely, cell wall signalling connects the state of cell wall homeostasis to the BR receptor complex and modulates BR activity. Here we report that both pectin-triggered cell wall signalling and impaired BR signalling result in altered cell wall orientation in the Arabidopsis root meristem. BR-induced defects in the orientation of newly placed walls are associated with aberrant localization of the cortical division zone but with normal specification of its positioning. Tissue- specific perturbations of BR signalling revealed that the cellular malfunction is unrelated to previously described whole organ growth defects. Thus, tissue type separates the pleiotropic effects of cell wall/BR signals and highlights their importance during cell wall placement.

The cells of all organisms are sugarcoated with polysaccharides, glycoproteins, and glycolipids. In multicellular organisms these polymers not only have a structural role in the organization of tissues and organs, but also have signaling activities. In the animal extracellular matrix for instance, the negatively charged hyaluronic acid (HA) polymers contribute to morphogenesis through mechanosensing, whereas HA fragments that are released from the polymer under certain conditions trigger inflammatory responses and angiogenesis (1). In plants, pectins are the major charged polysaccharides in the cell wall and thus can be considered the equivalent of HA. In addition, pectin-derived oligosaccharides released from the host cell wall during pathogen infection have been shown to trigger immune responses (2⇓–4). Gallego-Giraldo et al. (5) in PNAS now provide evidence for a role of pectin oligosaccharide signaling also in the response of plant cells to cell wall perturbation. The study is part of a larger effort of Gallego-Giraldo et al. (5) to tailor plant biomass for improved digestibility (for animal feed) or conversion into fermentable sugars (saccharification, to produce second-generation biofuels and chemicals) (6). Plants, in contrast to animals, contain lignins in their extracellular matrix, besides polysaccharides and glycoproteins. Lignins consist of oxidatively cross-linked phenolic molecules, which form a hydrophobic network attached to the polysaccharides. Previous studies by Gallego-Giraldo et al. and others have shown that lignins are a major obstacle for biomass digestibility/saccharification, mainly because they limit the access of cell … [↵][1]1To whom correspondence may be addressed. Email: hermanus.hofte{at}inra.fr. [1]: #xref-corresp-1-1

Growth of etiolated Arabidopsis hypocotyls is biphasic. During the first phase, cells elongate slowly and synchronously. At 48 h after imbibition, cells at the hypocotyl base accelerate their growth. Subsequently, this rapid elongation propagates through the hypocotyl from base to top. It is largely unclear what regulates the switch from slow to fast elongation. Reverse genetics-based screening for hypocotyl phenotypes identified three independent mutant lines of At1g70990, a short extensin (EXT) family protein that we named EXT33, with shorter etiolated hypocotyls during the slow elongation phase. However, at 72 h after imbibition, these dark-grown mutant hypocotyls start to elongate faster than the wild type (WT). As a result, fully mature 8-day-old dark-grown hypocotyls were significantly longer than WTs. Mutant roots showed no growth phenotype. In line with these results, analysis of native promoter-driven transcriptional fusion lines revealed that, in dark-grown hypocotyls, expression occurred in the epidermis and cortex and that it was strongest in the growing part. Confocal and spinning disk microscopy on C-terminal protein-GFP fusion lines localized the EXT33-protein to the ER and cell wall. Fourier-transform infrared microspectroscopy identified subtle changes in cell wall composition between WT and the mutant, reflecting altered cell wall biomechanics measured by constant load extensometry. Our results indicate that the EXT33 short EXT family protein is required during the first phase of dark-grown hypocotyl elongation and that it regulates the moment and extent of the growth acceleration by modulating cell wall extensibility.

International audience; Despite an ever-increasing interest for the use of pectin-derived oligogalacturonides (OGs) as biological control agents in agriculture, very little information exists-mainly for technical reasons-on the nature and activity of the OGs that accumulate during pathogen infection. Here we developed a sensitive OG profiling method, which revealed unsuspected features of the OGs generated during infection of Arabidopsis thaliana with the fungus Botrytis cinerea. Indeed, in contrast to previous reports, most OGs were acetyl-and methylesterified, and 80% of them were produced by fungal pectin lyases, not by polygalacturonases. Polygalacturonase products did not accumulate as larger size OGs but were converted into oxidized GalA dimers. Finally, the comparison of the OGs and transcriptomes of leaves infected with B. cinerea mutants with reduced pectinolytic activity but with decreased or increased virulence, respectively, identified candidate OG elicitors. In conclusion, OG analysis provides insights into the enzymatic arms race between plant and pathogen and facilitates the identification of defense elicitors. oligogalacturonides | plant-pathogen interaction | Arabidopsis thaliana | Botrytis cinerea | pectin lyase T he cell wall forms the first line of defense in the interaction of plants with their microbial environment. Cell walls consist of complex polysaccharide networks, which combine multiple functional properties such as strength (to resist turgor pressure), ex-tensibility (to allow growth), as well as protection against microbial attack. Such protection involves so called "basal immunity," which is triggered upon recognition of pathogen-associated molecular patterns but also of plant-derived molecules associated with infection, referred to as damage-associated molecular patterns (DAMPs). The latter include cell wall-derived oligosaccharides with elicitor activity (also called oligosaccharins) (1) that are produced during infection with microbes and, in particular, necrotrophic bacteria and fungi, which feed on cell walls and have large arsenals of cell wall-degrading enzymes (2). A major source of cell wall-derived DAMPs are pectins. The pectic polymer, homogalacturonan (HG) for example, represents in Arabidopsis 20% of the wall of growing cells. HG is a linear polymer of α-1-4-linked galacturonic acid (GalA) residues secreted in a methylesterified (on C6) and often acetylesterified (on C2 and/or C3) form (3), which can be enzymatically deacetylesterified by pectin acetylesterases and demethylesterified by pectin methyl-esterases (PMEs) in muro (4). Demethylesterification exposes the polymer to degradation by polygalacturonases (PGs) and pectate lyases (PLs, that cleave unmethylesterified HG by β-elimination, thus generating an unsaturated bond at the nonreducing end), which can be of plant or fungal origin. HG demethylesterification plays a key role in the control of cell wall rheology underlying plant growth (5). In addition, HG turnover generates oligogalacturonides (OGs)

A major challenge in plant systems biology is the development of robust, predictive multiscale models for organ growth. In this context it is important to bridge the gap between the, rather well-documented molecular scale and the organ scale by providing quantitative methods to study within-organ growth patterns. Here, we describe a simple method for the analysis of the evolution of growth patterns within rod-shaped organs that does not require adding markers at the organ surface. The method allows for the simultaneous analysis of root and hypocotyl growth, provides spatio-temporal information on curvature, growth anisotropy and relative elemental growth rate and can cope with complex organ movements. We demonstrate the performance of the method by documenting previously unsuspected complex growth patterns within the growing hypocotyl of the model species Arabidopsis thaliana during normal growth, after treatment with a growth-inhibiting drug or in a mechano-sensing mutant. The method is freely available as an intuitive and user-friendly Matlab application called KymoRod.

International audience; Somatic polyploidy caused by endoreplication is observed in arthropods, molluscs, and vertebrates but is especially prominent in higher plants, where it has been postulated to be essential for cell growth and fate maintenance. However, a comprehensive understanding of the physiological significance of plant endopolyploidy has remained elusive. Here, we modeled and experimentally verified a high-resolution DNA endoploidy map of the developing Arabidopsis thaliana root, revealing a remarkable spatiotemporal control of DNA endoploidy levels across tissues. Fitting of a simplified model to publicly available data sets profiling root gene expression under various environmental stress conditions suggested that this root endoploidy patterning may be stress-responsive. Furthermore, cellular and transcriptomic analyses revealed that inhibition of endoreplication onset alters the nuclear-to-cellular volume ratio and the expression of cell wall-modifying genes, in correlation with the appearance of cell structural changes. Our data indicate that endopolyploidy might serve to coordinate cell expansion with structural stability and that spatiotemporal endoreplication pattern changes may buffer for stress conditions, which may explain the widespread occurrence of the endocycle in plant species growing in extreme or variable environments.

Understanding how developmental and environmental signals control plant cell expansion requires an intimate knowledge of the architecture of the primary cell wall and the chemo-rheological processes that underlie cell wall relaxation. In this review I discuss recent findings that reveal a more prominent role than previously suspected for covalent bonds and pectin cross-links in primary cell wall architecture. In addition, genetic studies have uncovered a role for receptor kinases in the control of cell wall homeostasis in growing cells. The emerging view is that, upon cell wall disruption, compensatory changes are induced in the cell wall through the interplay between the brassinosteroid signaling module, which positively regulates wall extensibility and receptor kinases of the CrRLKL1 family, which may act as negative regulators of cell wall stiffness. These findings lift the tip of the veil of a complex signaling network allowing normal homeostasis in walls of growing cells but also crisis management under stress conditions.

Plants are able to generate large leaf surfaces that act as two-dimensional solar panels with a minimum investment in building material, thanks to a hydrostatic skeleton. This requires high intracellular pressures (up to 1 MPa), which depend on the presence of strong cell walls. The walls of growing cells (also called primary walls), are remarkably able to reconcile extreme tensile strength (up to 100 MPa) with the extensibility necessary for growth. All walled organisms are confronted with this dilemma - the need to balance strength and extensibility - and bacteria, fungi and plants have evolved independent solutions to cope. In this Primer, we discuss how plant cells have solved this problem, allowing them to support often very large increases in volume and to develop a broad variety of shapes (Figure 1A,B,D). This shape variation reflects the targeted deposition of wall material combined with local variations in cell-wall extensibility, processes that remain incompletely understood. Once the cell has reached its final size, it can lay down secondary wall layers, the composition and architecture of which are optimized to exert specific functions in different cell types (Figure 1E-G). Such functions include: providing mechanical support, for instance, for fibre cells in tree trunks or grass internodes; impermeabilising and strengthening vascular tissue to resist the negative pressure of the transpiration stream; increasing the surface area of the plasma membrane to facilitate solute exchange between cells (Figure 1C); or allowing important elastic deformation, for instance, to support the opening and closing of stomates. Specialized secondary walls, such as those constituting seed mucilage, are stored in a dehydrated form in seedcoat epidermis cells and show rapid swelling upon hydration of the seed. Other walls, in particular in reserve tissues, can accommodate large amounts of storage polysaccharides, which can be easily mobilized as a carbon source. Here we will discuss some general principles underlying wall architecture and wall growth that have emerged from recent studies, as well as future questions for investigation (Box 1).

International audience; hree types of treatments of miscanthus were performed, alkali, silanization and the combination of both. There is a direct inverse relation between the amount of sugar-containing molecules extracted from miscanthus and the mechanical strength of the concrete blocks. The use of alkali treated stems as fillers increased remarkably the strength of the blocks, and this was even higher when the alkali treated fibers were coated with silica, with a compression strength of 11 MPa compared with an initial 2.2 MPa. The use of alkali-treated stems result in a faster cement hydration, compared to untreated ones suggesting that specific sugars or other components released from the alkali-treated plant may favor cement hydration. A remarkable decrease of the cellulose and xylose content is observed for the miscanthus pieces after being soaked in a cement-water-sand mixture, these two molecules being adsorbed on cement particles

The brassinosteroid (BR) signaling module is a central regulator of plant morphogenesis, as indicated by the large number of BR-responsive cell wall-related genes and the severe growth defects of BR mutants. Despite a detailed knowledge of the signaling components, the logic of this auto-/paracrine signaling module in growth control remains poorly understood. Recently, extensive cross-talk with other signaling pathways has been shown, suggesting that the outputs of BR signaling, such as gene-expression changes, are subject to complex control mechanisms. We previously provided evidence for a role of BR signaling in a feedback loop controlling the integrity of the cell wall. Here, we identify the first dedicated component of this feedback loop: a receptor-like protein (RLP44), which is essential for the compensatory triggering of BR signaling upon inhibition of pectin de-methylesterification in the cell wall. RLP44 is required for normal growth and stress responses and connects with the BR signaling pathway, presumably through a direct interaction with the regulatory receptor-like kinase BAK1. These findings corroborate a role for BR in controlling the sensitivity of a feedback signaling module involved in maintaining the physico-chemical homeostasis of the cell wall during cell expansion.

While Brachypodium distachyon (Brachypodium) is an emerging model for grasses, no expression atlas or gene coexpression network is available. Such tools are of high importance to provide insights into the function of Brachypodium genes. We present a detailed Brachypodium expression atlas, capturing gene expression in its major organs at different developmental stages. The data were integrated into a large-scale coexpression database ( www.gene2function.de), enabling identification of duplicated pathways and conserved processes across 10 plant species, thus allowing genome-wide inference of gene function. We highlight the importance of the atlas and the platform through the identification of duplicated cell wall modules, and show that a lignin biosynthesis module is conserved across angiosperms. We identified and functionally characterised a putative ferulate 5-hydroxylase gene through overexpression of it in Brachypodium, which resulted in an increase in lignin syringyl units and reduced lignin content of mature stems, and led to improved saccharification of the stem biomass. Our Brachypodium expression atlas thus provides a powerful resource to reveal functionally related genes, which may advance our understanding of important biological processes in grasses.

Antagonistic growth effects of two T-DNA alleles in THESEUS1 correlate with the expression of a predicted functional THE1 protein lacking the kinase domain and antisense transcripts, respectively., Perturbation of cellulose synthesis in plants triggers stress responses, including growth retardation, mediated by the cell wall integrity-sensing receptor-like kinase (RLK) THESEUS1 (THE1). The analysis of two alleles carrying T-DNA insertions at comparable positions has led to conflicting conclusions concerning the impact of THE1 signaling on growth. Here we confirm that, unlike the1-3 and other the1 alleles in which cellular responses to genetic or pharmacological inhibition of cellulose synthesis are attenuated, the1-4 showed enhanced responses, including growth inhibition, ectopic lignification, and stress gene expression. Both the1-3 and the1-4 express a transcript encoding a predicted membrane-associated truncated protein lacking the kinase domain. However, the1-3, in contrast to the1-4, strongly expresses antisense transcripts, which are expected to prevent the expression of the truncated protein as suggested by the genetic interactions between the two alleles. Seedlings overexpressing such a truncated protein react to isoxaben treatment similarly to the1-4 and the full-length THE overexpressor. We conclude that the1-4 is a hypermorphic allele; that THE1 signaling upon cell wall damage has a negative impact on cell expansion; and that caution is required when interpreting the phenotypic effects of T-DNA insertions in RLK genes.

Plants actively perceive and respond to perturbations in their cell walls which arise during growth, biotic and abiotic stresses. However, few components involved in plant cell wall integrity sensing have been described to date. Using a reverse-genetic approach, we identified the Arabidopsis thaliana leucine-rich repeat receptor kinase MIK2 as an important regulator of cell wall damage responses triggered upon cellulose biosynthesis inhibition. Indeed, loss-of-function mik2 alleles are strongly affected in immune marker gene expression, jasmonic acid production and lignin deposition. MIK2 has both overlapping and distinct functions with THE1, a malectin-like receptor kinase previously proposed as cell wall integrity sensor. In addition, mik2 mutant plants exhibit enhanced leftward root skewing when grown on vertical plates. Notably, natural variation in MIK2 (also named LRR-KISS) has been correlated recently to mild salt stress tolerance, which we could confirm using our insertional alleles. Strikingly, both the increased root skewing and salt stress sensitivity phenotypes observed in the mik2 mutant are dependent on THE1. Finally, we found that MIK2 is required for resistance to the fungal root pathogen Fusarium oxysporum. Together, our data identify MIK2 as a novel component in cell wall integrity sensing and suggest that MIK2 is a nexus linking cell wall integrity sensing to growth and environmental cues., Author summary Plants are constantly exposed to external stresses of biotic and abiotic nature, as well as internal stresses, resulting from growth and mechanical tension. Feedback information about the integrity of the cell wall can enable the plant to perceive such stresses, and respond adequately. Plants are known to perceive signals from their environment through receptor kinases at the plant cell surface. Here, we reveal that the Arabidopsis thaliana receptor kinase MIK2 regulates responses to cell wall perturbation. Moreover, we find that MIK2 controls root growth angle, modulates cell wall structure in the root tip, contributes to salt stress tolerance, and is required for resistance against a root-infecting pathogen. Our data suggest that MIK2 is involved in sensing cell wall perturbations in plants, whereby it allows the plant to cope with a diverse range of environmental stresses. These data provide an important step forward in our understanding of the mechanisms plants deploy to sense internal and external danger.

Because the plant cell wall provides the first line of defense against biotic and abiotic assaults, its functional integrity needs to be maintained under stress conditions. Through a phenotype-based compound screening approach, we identified a novel cellulose synthase inhibitor, designated C17. C17 administration depletes cellulose synthase complexes from the plasma membrane in Arabidopsis thaliana, resulting in anisotropic cell elongation and a weak cell wall. Surprisingly, in addition to mutations in CELLULOSE SYNTHASE1 (CESA1) and CESA3, a forward genetic screen identified two independent defective genes encoding pentatricopeptide repeat (PPR)-like proteins (CELL WALL MAINTAINER1 [CWM1] and CWM2) as conferring tolerance to C17. Functional analysis revealed that mutations in these PPR proteins resulted in defective cytochrome c maturation and activation of mitochondrial retrograde signaling, as evidenced by the induction of an alternative oxidase. These mitochondrial perturbations increased tolerance to cell wall damage induced by cellulose deficiency. Likewise, administration of antimycin A, an inhibitor of mitochondrial complex III, resulted in tolerance toward C17. The C17 tolerance of cwm2 was partially lost upon depletion of the mitochondrial retrograde regulator ANAC017, demonstrating that ANAC017 links mitochondrial dysfunction with the cell wall. In view of mitochondria being a major target of a variety of stresses, our data indicate that plant cells might modulate mitochondrial activity to maintain a functional cell wall when subjected to stresses.

SummaryBrassinosteroid (BR) signaling is required for normal plant growth as shown by the dwarf phenotype of loss-of-function BR biosynthetic or perception mutants. Despite a detailed understanding of the BR signaling network [1–3], it is not clear how exactly BRs control growth. For instance, genetic sector analysis shows that BRs, in contrast to most other growth regulators, act locally, presumably in an autocrine and/or paracrine mode, suggesting that they have some role in feedback regulation [4, 5]. Here, we show that at least one role for BRs in growth control is to ensure pectin-dependent cell wall homeostasis. Pectins are complex block cell wall polymers [6], which can be modified in the wall by the enzyme pectin methylesterase (PME) [7]. Genetic or pharmacological interference with PME activity causes dramatic changes in growth behavior, which are primarily the result of the activation of the BR signaling pathway. We propose that this activation of BR signaling is part of a compensatory response, which protects the plant against the loss of cell wall integrity caused by the imbalance in pectin modification. Thus, feedback signaling from the cell wall is integrated by the BR signaling module to ensure homeostasis of cell wall biosynthesis and remodeling.

SummaryTissue mechanics have been shown to play a key role in the regulation of morphogenesis in animals [1–4] and may have an equally important role in plants [5–9]. The aerial organs of plants are formed at the shoot apical meristem following a specific phyllotactic pattern [10]. The initiation of an organ from the meristem requires a highly localized irreversible surface deformation, which depends on the demethylesterification of cell wall pectins [11]. Here, we used atomic force microscopy (AFM) to investigate whether these chemical changes lead to changes in tissue mechanics. By mapping the viscoelasticity and elasticity in living meristems, we observed increases in tissue elasticity, correlated with pectin demethylesterification, in primordia and at the site of incipient organs. Measurements of tissue elasticity at various depths showed that, at the site of incipient primordia, the first increases occurred in subepidermal tissues. The results support the following causal sequence of events: (1) demethylesterification of pectin is triggered in subepidermal tissue layers, (2) this contributes to an increase in elasticity of these layers—the first observable mechanical event in organ initiation, and (3) the process propagates to the epidermis during the outgrowth of the organ.

International audience; The growth of plants, like that of other walled organisms, depends on the ability of the cell wall to yield without losing its integrity. In this context, plant cells can sense the perturbation of their walls and trigger adaptive modifications in cell wall polymer interactions. Catharanthus roseus receptor-like kinase 1-like (CrRLK1L) THESEUS1 (THE1) was previously shown in Arabidopsis to trigger growth inhibition and defense responses upon perturbation of the cell wall, but so far, neither the ligand nor the role of the receptor in normal development was known. Here, we report that THE1 is a receptor for the peptide rapid alkalinization factor (RALF) 34 and that this signaling module has a role in the fine-tuning of lateral root initiation. We also show that RALF34-THE1 signaling depends, at least for some responses, on FERONIA (FER), another RALF receptor involved in a variety of processes, including immune signaling, mechanosensing, and reproduction [1]. Together, the results show that RALF34 and THE1 are part of a signaling network that integrates information on the integrity of the cell wall with the coordination of normal morphogenesis.

The growth of plant cells involves a constant adjustment of synthesis and rearrangement of cell wall polymers. Recently, three plasma membrane-bound receptor kinases related to CrRLK1 have been shown to be involved in the negative control of cell growth in different contexts. THESEUS1 is activated in mutants deficient for cellulose and may act as a cell wall integrity sensor inhibiting cell elongation. FERONIA is polarly localized in synergid cells of the female gametophyte and is required for growth cessation of compatible pollen tubes and subsequent delivery of sperm cells. AmRLK is involved in the control of the polar conical outgrowth of epidermal cells of Antirrhinum petals. The conservation of both extracellular and kinase domains suggests that the three receptors bind to related ligands and have similar cellular outputs, which may involve the production of reactive oxygen species.

Plants, like yeast, have the ability to monitor alterations in the cell wall architecture that occur during normal growth or in changing environments and to trigger compensatory changes in the cell wall. We discuss how recent advances in our understanding of the cell wall architecture provide new insights into the role of cell wall integrity sensing in growth control. Next we review the properties of membrane receptor-like kinases that have roles in pH control, mechano-sensing and reactive oxygen species accumulation in growing cells and which may be the plant equivalents of the yeast cell wall integrity (CWI) sensors. Finally, we discuss recent findings showing an increasing role for CWI signaling in plant immunity and the adaptation to changes in the ionic environment of plant cells.

Glycosyl, or glycoside, hydrolases (GHs) comprise a structurally diverse group of enzymes that hydrolyze glycosidic bonds between carbohydrates, or between carbohydrates and other noncarbohydrate moieties, and that collectively exhibit a wide range of substrate specificities. GH enzymes from across

Summary Background A major challenge is to understand how the walls of expanding plant cells are correctly assembled and remodeled, often in the presence of wall-degrading micro-organisms. Plant cells, like yeast, react to cell-wall perturbations as shown by changes in gene expression, accumulation of ectopic lignin, and growth arrest caused by the inhibition of cellulose synthesis. Results We have identified a plasma-membrane-bound receptor-like kinase (THESEUS1), which is present in elongating cells. Mutations in THE1 and overexpression of a functional THE1-GFP fusion protein did not affect wild-type (WT) plants but respectively attenuated and enhanced growth inhibition and ectopic lignification in seedlings mutated in cellulose synthase CESA6 without influencing the cellulose deficiency. A T-DNA insertion mutant for THE1 also attenuated the growth defect and ectopic-lignin production in other but not all cellulose-deficient mutants. The deregulation of a small number of genes in cesA6 mutants depended on the presence of THE1. Some of these genes are involved in pathogen defense, in wall crosslinking, or in protecting the cell against reactive oxygen species. Conclusions The results show that THE1 mediates the response of growing plant cells to the perturbation of cellulose synthesis and may act as a cell-wall-integrity sensor.

Pectins are a family of complex cell-wall polysaccharides, the biosynthesis of which remains poorly understood. We identified dwarf mutants with reduced cell adhesion at a novel locus, QUASIMODO2 (QUA2). qua2-1 showed a 50% reduction in homogalacturonan (HG) content compared with the wild type, without affecting other cell-wall polysaccharides. The remaining HG in qua2-1 showed an unaltered degree of methylesterification. Positional cloning and GFP fusions showed that QUA2, consistent with a role in HG synthesis, encodes a Golgi-localized protein. In contrast to QUA1, another Golgi-localized protein required for HG-synthesis, QUA2 does not show sequence similarity to glycosyltransferases, but instead contains a putative methyltransferase (MT) domain. The Arabidopsis genome encodes 29 QUA2-related proteins. Interestingly, the transcript profiles of QUA1 and QUA2 are correlated and other pairs of QUA1 and QUA2 homologues with correlated transcript profiles can be identified. Together, the results lead to the hypothesis that QUA2 is a pectin MT, and that polymerization and methylation of homogalacturonan are interdependent reactions.

Early ViewTechnical advance; The localization of proteins in specific domains or compartments in the 3D cellular space is essential for many fundamental processes in eukaryotic cells. Deciphering spatial organization principles within cells is a challenging task, in particular because of the large morphological variations between individual cells. We present here an approach for normalizing variations in cell morphology and for statistically analyzing spatial distributions of intracellular compartments from collections of 3D images. The method relies on the processing and analysis of 3D geometrical models that are generated from image stacks and that are used to build representations at progressively increasing levels of integration, ultimately revealing statistical significant traits of spatial distributions. To make this methodology widely available to end-users, we implemented our algorithmic pipeline into a user-friendly, multi-platform, and freely available software. To validate our approach, we generated 3D statistical maps of endomembrane compartments at subcellular resolution within an average epidermal root cell from collections of image stacks. This revealed unsuspected polar distribution patterns of organelles that were not detectable in individual images. By reversing the classical ‘measure-then-average’ paradigm, one major benefit of the proposed strategy is the production and display of statistical 3D representations of spatial organizations, thus fully preserving the spatial dimension of image data and at the same time allowing their integration over individual observations. The approach and software are generic and should be of general interest for experimental and modeling studies of spatial organizations at multiple scales (subcellular, cellular, tissular) in biological systems.

Summary A mutant called defective glycosylation1-1 (dgl1-1) was identified in Arabidopsis based on a growth defect of the dark-grown hypocotyl and an abnormal composition of the non-cellulosic cell wall polysaccharides. dgl1-1 is altered in a protein ortholog of human OST48 or yeast WBP1, an essential protein subunit of the oligosaccharyltransferase (OST) complex, which is responsible for the transfer in the ER of the N-linked glycan precursor onto Asn residues of candidate proteins. Consistent with the known function of the OST complex in eukaryotes, the dgl1-1 mutation led to a reduced N-linked glycosylation of the ER-resident protein disulfide isomerase. A second more severe mutant (dgl1-2) was embryo-lethal. Microscopic analysis of dgl1-1 revealed developmental defects including reduced cell elongation and the collapse and differentiation defects of cells in the central cylinder. These defects were accompanied by changes in the non-cellulosic polysaccharide composition, including the accumulation of ectopic callose. Interestingly, in contrast to other dwarf mutants that are altered in early steps of the N-glycan processing, dgl1-1 did not exhibit a cellulose deficiency. Together, these results confirm the role of DGL1 in N-linked glycosylation, cell growth and differentiation in plants.

Cellulose-deficient Arabidopsis mutants were identified using FT-IR microspectroscopy. The study of these mutants not only led to the identification of actors in cellulose synthesis, but also provided insights in the organization of the hexameric terminal complex from CESA mutants and identified unsuspected accessory proteins with so far unknown roles in the synthesis and/or assembly of cellulose microfibrils. Finally, mutant analysis established a role for protein glycosylation in cellulose synthesis and provided new perspectives on the developmental regulation of cell wall synthesis and the role that cellulose synthesis plays in the control of cell elongation.

A central problem in plant biology is how cell expansion is coordinated with wall synthesis. We have studied growth and wall deposition in epidermal cells of dark-grown Arabidopsis hypocotyls. Cells elongated in a biphasic pattern, slowly first and rapidly thereafter. The growth acceleration was initiated at the hypocotyl base and propagated acropetally. Using transmission and scanning electron microscopy, we analyzed walls in slowly and rapidly growing cells in 4-d-old dark-grown seedlings. We observed thick walls in slowly growing cells and thin walls in rapidly growing cells, which indicates that the rate of cell wall synthesis was not coupled to the cell elongation rate. The thick walls showed a polylamellated architecture, whereas polysaccharides in thin walls were axially oriented. Interestingly, innermost cellulose microfibrils were transversely oriented in both slowly and rapidly growing cells. This suggested that transversely deposited microfibrils reoriented in deeper layers of the expanding wall. No growth acceleration, only slow growth, was observed in the cellulose synthase mutant cesA6prc1-1 or in seedlings, which had been treated with the cellulose synthesis inhibitor isoxaben. In these seedlings, innermost microfibrils were transversely oriented and not randomized as has been reported for other cellulose-deficient mutants or following treatment with dichlorobenzonitrile. Interestingly, isoxaben treatment after the initiation of the growth acceleration in the hypocotyl did not affect subsequent cell elongation. Together, these results show that rapid cell elongation, which involves extensive remodeling of the cell wall polymer network, depends on normal cellulose deposition during the slow growth phase.

The ability of cells to perceive changes in the composition and mechanical properties of their cell wall is crucial for plants to achieve coordinated growth and development. Evidence is accumulating to show that the plant cell wall, like its yeast counterpart, is capable of triggering multiple signalling pathways. The components of the cell wall that are responsible for initiating these signal responses remain unknown; however, recent technological advances in cell wall analysis may now facilitate the identification of these components and accelerate the characterisation of changes that occur in cell wall mutants.

The fourth author’s name is spelled incorrectly. The correct name is: Claire L. Alvim-Kamei. The correct citation is: Mansoori N, Timmers J, Desprez T, Alvim-Kamei CL, Dees DCT, Vincken J-P, et al. (2014) KORRIGAN1 Interacts Specifically with Integral Components of the Cellulose Synthase Machinery. PLoS ONE 9(11): e112387. doi:10.1371/journal.pone.0112387

in press; Complex shapes in biology depend on the ability of cells to shift from isotropic to anisotropic growth during development. In plants, this growth symmetry breaking reflects changes in the extensibility of the cell walls. The textbook view is that the direction of turgor-driven cell expansion depends on the cortical microtubule (CMT)-mediated orientation of cellulose microfibrils [1, 2]. Here, we show that this view is incomplete at best. We used atomic force microscopy (AFM) to study changes in cell-wall mechanics associated with growth symmetry breaking within the hypocotyl epidermis. We show that, first, growth symmetry breaking is preceded by an asymmetric loosening of longitudinal, as compared to transverse, anticlinal walls, in the absence of a change in CMT orientation. Second, this wall loosening is triggered by the selective de-methylesterification of cell-wall pectin in longitudinal walls, and, third, the resultant mechanical asymmetry is required for the growth symmetry breaking. Indeed, preventing or promoting pectin de-methylesterification, respectively, increased or decreased the stiffness of all the cell walls, but in both cases reduced the growth anisotropy. Finally, we show that the subsequent CMT reorientation contributes to the consolidation of the growth axis but is not required for the growth symmetry breaking. We conclude that growth symmetry breaking is controlled at a cellular scale by bipolar pectin de-methylesterification, rather than by the cellulose-dependent mechanical anisotropy of the cell walls themselves. Such a cell asymmetry-driven mechanism is comparable to that underlying tip growth in plants [3] but also anisotropic cell growth in animal cells [4].

Recent studies have highlighted the involvement of membrane-anchored endo-beta-1,4-glucanases in cellulose biosynthesis in plants, suggesting that there are parallels with Agrobacterium tumefaciens and other bacteria which also require endo-beta-1,4-glucanases for cellulose synthesis. This review summarises recent literature on endo-beta-1,4-glucanases and their role in plant development and addresses the possible functions of membrane-anchored isoforms in the synthesis of cellulose.

International audience; Pectins are a highly complex family of cell wall polysaccharides. As a result of a lack of specific mutants, it has been difficult to study the biosynthesis of pectins and their role in vivo. We have isolated two allelic mutants, named quasimodo1 (qua1-1 and qua1-2), that are dwarfed and show reduced cell adhesion. Mutant cell walls showed a 25% reduction in galacturonic acid levels compared with the wild type, indicating reduced pectin content, whereas neutral sugars remained unchanged. Immersion immunofluorescence with the JIM5 and JIM7 monoclonal antibodies that recognize homogalacturonan epitopes revealed less labeling of mutant roots compared with the wild type. Both mutants carry a T-DNA insertion in a gene (QUA1) that encodes a putative membrane-bound glycosyltransferase of family 8. We present evidence for the possible involvement of a glycosyltransferase of this family in the synthesis of pectic polysaccharides, suggesting that other members of this large multigene family in Arabidopsis also may be important for pectin biosynthesis. The mutant phenotype is consistent with a central role for pectins in cell adhesion.

The cell wall is the major limiting factor for plant growth. Wall extension is thought to result from the loosening of its structure. However, it is not known how this is coordinated with wall synthesis. We have identified two novel allelic cellulose-deficient dwarf mutants, kobito1-1 and kobito1-2 (kob1-1 and kob1-2). The cellulose deficiency was confirmed by the direct observation of microfibrils in most recent wall layers of elongating root cells. In contrast to the wild type, which showed transversely oriented parallel microfibrils, kob1 microfibrils were randomized and occluded by a layer of pectic material. No such changes were observed in another dwarf mutant, pom1, suggesting that the cellulose defect in kob1 is not an indirect result of the reduced cell elongation. Interestingly, in the meristematic zone of kob1 roots, microfibrils appeared unaltered compared with the wild type, suggesting a role for KOB1 preferentially in rapidly elongating cells. KOB1 was cloned and encodes a novel, highly conserved, plant-specific protein that is plasma membrane bound, as shown with a green fluorescent protein–KOB1 fusion protein. KOB1 mRNA was present in all organs investigated, and its overexpression did not cause visible phenotypic changes. KOB1 may be part of the cellulose synthesis machinery in elongating cells, or it may play a role in the coordination between cell elongation and cellulose synthesis.